Diagnóstico imuno-histoquímico e caracterização anatomopatológica de Mycoplasma gallisepticum em galinhas de subsistência

A micoplasmose aviária é causada por bactérias da família Mycoplasmataceae. Mycoplasma gallisepticum (MG) é a espécie mais patogênica e que tem a maior importância econômica para a produção avícola. Este estudo teve por objetivo utilizar a técnica de imuno-histoquímica (IHQ) como método de diagnóstico da infecção por MG em aves. No presente relato são descritos dois surtos de micoplasmose por MG em galinhas de subsistência. Clinicamente as aves apresentaram prostração, hiporexia, dificuldade respiratória, secreção nasal e ocular. Na necropsia foram observados secreção serosa, edema e deposição de cáseo em conjuntiva (7/10) e seios nasais (4/10), sacos aéreos espessados com espuma e cáseo (6/10); traqueia difusamente avermelhada (4/10); pulmões com pontos esbranquiçados de 0,5cm (2/10); e saco pericárdico com deposição de fibrina (2/10). No exame histopatológico foram evidenciados traqueíte (10/10), sinusite (5/5) e conjuntivite (3/4) hiperplásica linfoplasmocitária aguda; broncopneumonia fibrinonecrótica (5/10); pericardite fibrinosa aguda (2/10); e aerossaculite fibrinonecrótica (1/1). No exame de IHQ anti-MG foi evidenciada marcação na superfície extracelular dos cílios e/ou topo do epitélio da traqueia (10/10), brônquios (5/10) e seios nasais (4/5). Em sete dos dez casos analisados foi detectada a presença de MG por PCR em tempo real realizado a partir de amostras de suabe traqueal. A técnica de IHQ anti-MG utilizada como método de diagnóstico apresentou boa concordância com os sinais clínicos, as lesões histopatológicas e os resultados de PCR em tempo real.

Occurrence of Mycoplasma synoviae on commercial poultry farms of Pernambuco, Brazil

The state of Pernambuco is the largest producer of eggs in the North and Northeast of Brazil and second one in the broiler production. Mycoplasmas are important avian pathogens, which cause respiratory and joint diseases that result in large economic losses. The aim of the present study was to investigate the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in broilers and commercial laying hens in the state of Pernambuco, Brazil. Tracheal fragments were analyzed from 55 healthy broilers, 35 broilers with respiratory signs and 30 commercial laying hens with respiratory signs, from 24 commercial poultry farms, each sample was composed of a pool of five birds. The bacteriological exam, PCR and nested PCR were used for the detection of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). All samples were negative in bacteriological isolation. In the PCR analyses, seven samples from birds with respiratory signs were positive for MS and one was positive for MG, the latter of which was confirmed as the MG-F vaccine strain. The occurrence of MS in chickens with respiratory signs may indicate inadequate sanitary management on poultry farms, favoring the propagation of mycoplasmosis.

Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD) of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR) demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%), and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks.

Occurrence and risk factors assessment associated with Mycoplasma gallisepticum (MG) infection in chickens in the semiarid region of Pernambuco, Brazil

Abstract: The aim of the present study was to assess the occurrence of Mycoplasma gallisepticum (MG) infection and risk factors of this disease in three hundred serum samples from on 23 familiar agricultural properties in the semiarid region of the state of Pernambuco, Brazil. ELISA was used to study antibodies anti-Mycoplasma gallisepticum. The univariate analysis (chi-squared test or Fischer's exact test) followed by multivariate analysis (logistic regression) were used to assess the risk factors with two variables: management and sanity of the poultry. It was detected a frequence of 53.33% (157/300) of the birds were positive for MG, with 100% foci. The risk factors confirmed by multivariate analysis, in the present study, were the presence of other poultry species on the property, including Numida meleagris (OR=2.22; p=0.005), parrots (OR=1.72; p=0.027), and of passerines (OR=1.88; p=0.007). These results showed that Mycoplasma gallisepticum infection is endemic among backyard poultry in the semiarid region of the state of Pernambuco. These birds could be a source of infection for other wild or domestic poultry. . This is the first report of the occurrence of avian mycoplasmosis in backyard poultry in the state of Pernambuco in northeastern Brazil. The risk factors identified should serve as a parameter for the health authorities to seek solutions related to controlling the disease.

Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells

Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 ± 8.6%) and low production of NO (4.7 ± 3.1 µM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 ± 9.1%; P<0.05) and higher NO production (48.5 ± 13 µM NO2-; P<0.05). Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 ± 2% (P<0.05) and NO production to 3 ± 4 µM NO2- (P<0.05). The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.

Detection of Mycoplasma pneumoniae and Chlamydia pneumoniae in ruptured atherosclerotic plaques

This paper reports what is apparently the first observation of Mycoplasma pneumoniae in association with Chlamydia pneumoniae in thrombosed ruptured atheromas. We performed electron microscopy and in situ hybridization in specimens from three patients who died of acute myocardial infarction. These patients had typical symptoms of acute ischemic syndrome. Mycoplasmas were present mainly in the lipid core of the ruptured thrombosed plaque. Vulnerable atheromas are rich in cholesterol and may favor the growth of mycoplasmas, the only microorganisms that require cholesterol for survival. We suggest that the association of Mycoplasma pneumoniae and Chlamydia pneumoniae may increase the virulence of these microorganisms, favoring proliferation, plaque inflammation and possibly plaque rupture.

Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

Implementación del diagnóstico de mycoplasma gallisepticum y mycoplasma synoviae mediante PCR-RFLP en aves de producción

UANL

Étude de la diversité génétique d’isolats québécois de Mycoplasma hyopneumoniae provenant d’infections simples ou mixtes et caractérisation de leur sensibilité aux antimicrobiens

Mycoplasma hyopneumoniae est l’agent causal de la pneumonie enzootique. On le retrouve dans plusieurs élevages de porcs à travers le monde. Même si ce micro-organisme est présent dans plusieurs troupeaux canadiens, peu d’informations sont présentement disponibles sur les isolats québécois. Un total de 160 poumons de porcs possédant des lésions de pneumonie ont été récupérés à l’abattoir, mis en culture et testés par PCR pour M. hyopneumoniae et Mycoplasma hyorhinis. D’autres pathogènes bactériens communs du porc et les virus du syndrome reproducteur et respiratoire porcin (VSRRP), de l’influenza et le circovirus porcin de type 2 (CVP2) ont été également testés. Quatre-vingt-dix pourcent des échantillons étaient positifs pour M. hyopneumoniae et 5.6% l’étaient seulement pour M. hyorhinis. Dans ces échantillons positifs pour M. hyopneumoniae, la concentration de ce mycoplasme variait de 1.17 x 105 à 3.37 x 109 génomes/mL. Vingt-cinq poumons positifs en culture ou par PCR en temps réel pour M. hyopneumoniae ont été sélectionnés, parmi ceux-ci 10 étaient en coinfection avec Pasteurella multocida, 12 avec Streptococcus suis, 9 avec CVP2 et 2 avec le VSRRP. Les analyses des nombres variables de répétitions en tandem à de multiples loci (MLVA) et PCR-polymorphisme de longueur de fragments de restriction (PCR-RFLP) de M. hyopneumoniae ont démontré une forte diversité des isolats de terrain. Par contre, il semble y avoir plus d’homogénéité à l’intérieur d’un même élevage. L’analyse MLVA a également démontré que près de la moitié des isolats possédaient moins de 55% d’homologie avec les souches vaccinales et de référence utilisées dans la présente étude. L’absence d’amplification du locus 1 de M. hyopneumoniae en MLVA a été significativement associée à une baisse de la concentration de bactérie et de la sévérité des lésions. Pour tous les isolats de M. hyopneumoniae, des concentrations minimales inhibitrices (CMI) de faibles à intermédiaires ont été obtenues envers tous les antimicrobiens testés. Les isolats possédant des CMI intermédiaires envers les tétracyclines, les macrolides et les lincosamides ont été testés pour la présence des gènes de résistance tetM, ermB et pour des mutations ponctuelles dans les gènes des protéines L4, L22 et de l’ARNr 23S. Aucun de ces gènes n’a été détecté mais la mutation ponctuelle G2057A a été identifiée. Cette mutation est responsable de la résistance intrinsèque de M. hyopneumoniae face aux macrolides à 14 carbones. Ces résultats indiquent qu’il ne semble pas y avoir de résistance acquise aux antimicrobiens parmi ces isolats. En conclusion, cette recherche a permis d’obtenir de nouvelles données scientifiques sur les isolats québécois de M. hyopneumoniae.

Excrétion nasale et réponse sérologique à Mycoplasma bovis chez les génisses de remplacement de 0 à 7 mois d'âge dans 4 troupeaux laitiers au Québec: Étude de cohortes

En Amérique du Nord, Mycoplasma bovis est le plus pathogène des mycoplasmes retrouvés chez les bovins. Les principales maladies qu’on lui associe (maladies respiratoires, mammites, arthrites septiques et otites moyennes et/ou internes) constituent un défi à l’industrie laitière à cause de la difficulté à les traiter et à les prévenir par une vaccination. L’objectif principal de ce projet était d’étudier l’excrétion nasale et la réponse sérologique à M. bovis chez les génisses de remplacement, entre la naissance et 7 mois d’âge, dans 4 troupeaux laitiers au Québec. Quatre-vingt-trois paires mère/génisse provenant de 4 cohortes de bovins laitiers étaient prélevées mensuellement (génisses : 0 à 7 mois ; mères : 0, 1 et 5 mois après vêlage). Écouvillons nasaux et échantillons de lait étaient analysés par culture bactériologique et par immunofluorescence indirecte. Les anticorps circulants étaient détectés par le test ELISA. À la naissance, la prévalence sérologique des génisses était supérieure à celle des mères (P = 0,01). La transmission de M. bovis aux génisses par le lait et par l’excrétion nasale des mères était faible. L’âge moyen (jour) d’une génisse à sa 1ère excrétion nasale et sa 1ère séroconversion à M. bovis était loin de la période néonatale: 77,5 ± 11,2 (n = 22) et 96,8 ± 7,4 (n = 36) respectivement. Conclusion, les vaches adultes n’ont constitué qu’une voie mineure de transmission de M. bovis aux génisses, la principale voie de transmission était fort probablement le contact direct ou indirect avec d’autres génisses excrétrices nasales de M. bovis.

Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, is present in swine herds worldwide. However, there is little information on strains infecting herds in Canada. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia originating from 48 different farms were recovered from two slaughterhouses and submitted for gross pathology. The pneumonic lesion scores ranged from 2% to 84%. Eighty nine percent of the lungs (143/160) were positive for M. hyopneumoniae by real-time PCR whereas 10% (16/160) and 8.8% (14/160) were positive by PCR for M. hyorhinis and M. flocculare, respectively. By culture, only 6% of the samples were positive for M. hyopneumoniae (10/160). Among the selected M. hyopneumoniae-positive lungs (n = 25), 9 lungs were co-infected with M. hyorhinis, 9 lungs with PCV2, 2 lungs with PRRSV, 12 lungs with S. suis and 10 lungs with P. multocida. MLVA and PCR-RFLP clustering of M. hyopneumoniae revealed that analyzed strains were distributed among three and five clusters respectively, regardless of severity of lesions, indicating that no cluster is associated with virulence. However, strains missing a specific MLVA locus showed significantly less severe lesions and lower numbers of bacteria. MLVA and PCR-RFLP analyses also showed a high diversity among field isolates of M. hyopneumoniae with a greater homogeneity within the same herd. Almost half of the field isolates presented less than 55% homology with selected vaccine and reference strains.

Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine.

Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 ± 9.6%) compared to the unvaccinated and challenged animals (45.8 ± 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 ± 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 ± 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn® MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines.

Modelling of control options for an outbreak of Mycoplasma gallisepticum in egg production: a decision support tool

Mycoplasma gallisepticum (MG) is a bacterium that causes respiratory disease in chickens, leading to reduced egg production. A dynamic simulation model was developed that can be used to assess the costs and benefits of control using antimicrobials or vaccination in caged or free range systems. The intended users are veterinarians and egg producers. A user interface is provided for input of flock specific parameters. The economic consequence of an MG outbreak is expressed as a reduction in expected egg output. The model predicts that either vaccination or microbial treatment can approximately halve potential losses from MG in some circumstances. Sensitivity analysis is used to test assumptions about infection rate and timing of an outbreak. Feedback from veterinarians points to the value of the model as a discussion tool with producers.